lentiCRISPR v2

CRISPR Human - Deletion SST3B

CRISPR Human - Deletion SST3B
lentiCRISPR v2 from Addgene

Protocol tips

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.
Protocol tips
There is no need to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

CRISPR Genetic Screens
Huh7.5.1 cells were stably transduced with lentiCas9-Blast and subsequently selected using blasticidin. Next, a total of 300 million Huh7.5.1 cells that constitutively express Cas9 were transduced with the lentiGuide-Puro from the GeCKO v2 library31 at a MOI of 0.3. Cells were selected with puromycin and pooled together. The CRISPR genetic screens were started 10 days post transduction. 60 million mutagenized cells for each library (A and B) were infected with DENV2 16681 (replicate 1 and 2) or DENV2 strain 429557 (replicate 3) using a MOI of 1, or with HCV JFH1 at a MOI of 0.3. Cytopathic effect was visible 2 days and 5 days post infection for DENV and HCV, respectively. Huh7.5.1 cells grow slower than HAP1 cells and clusters of resistant cells took longer to develop. The selected cells were harvested 16 days after infection. As uninfected reference we chose the unselected starting population because in these strong positive selection screens the selection pressure of the viral infections renders potential small growth differences of the mutagenized cells inconsequential. For both selected and uninfected control cells gDNA was isolated using a QIAamp DNA columns, and the inserted guideRNA sequences were amplified from the gDNA by flanking primers and prepared for next-generation sequencing. Resulting amplicons were sequenced on a MiSeq or NextSeq platform (Illumina) and the enrichment of each guideRNA was calculated by comparing the relative abundance in the selected and unselected population. RIGER analysis was performed on guide RNAs (with at least 10 reads) ranked by enrichment using the weighted sum statistical method32. Each CRISPR screen was performed in 3 replicates and the mean of the 3 RIGER scores was calculated.

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4 years ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

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